Question: "Shifted" copy number ratios coming from cnvkit?
gravatar for gil.hornung
3.7 years ago by
European Union
gil.hornung70 wrote:

I have data from three tumor samples from the same patient at different stages. The samples are derived from FFPE sections, and they underwent exome sequencing.

I used CNVkit to calculate the copy number ratio and segment the genome with parameters -m cbs -t 1e-4 --drop-low-coverage --drop-outliers 5.

I noticed that I have copy number gain or loss throughout the entire genome, and when I plot the distribution of the log2 copy number ratio I get two peaks, above and below log2==0 (I would expect many segments to have log2==0). Is it possible that the log2 values are shifted in some way from log2==0?

The plot: enter image description here

I am also attaching plots from BubbleTree enter image description here

somatic copy number cnvkit • 1.8k views
ADD COMMENTlink modified 3.7 years ago by Eric T.2.6k • written 3.7 years ago by gil.hornung70
gravatar for markus.riester
3.7 years ago by
markus.riester510 wrote:

When copy number ratios are calculated, you need to normalize tumor and normal for sequencing coverage differences. The problem is, however, that tumor cells can have more or less DNA than normal. Since you don't know the ploidy (average tumor copy number) when you calculate the log-ratios, you cannot adjust for ploidy differences at this step.

In your case, it looks like you have more losses than gains, so your tumor ploidy is below 2. By assuming a ploidy of 2 in the ratio calculation, the coverage is slightly overestimated and the peaks are right shifted. If the tumor ploidy would be > 2, then you would "remove" too many reads in the coverage normalization and the peaks would be left shifted.

See the ABSOLUTE paper for a nice explanation how tumor purity, ploidy and log-ratios relate to each other.

ADD COMMENTlink written 3.7 years ago by markus.riester510

@markus would like to know the basis of more losses than gains (from OP's post)? from right skew? Thanks

ADD REPLYlink modified 3.7 years ago • written 3.7 years ago by cpad011215k

Yeah, it's not 100% clear from the plots, because it looks like the log ratios are not weighted by segment size. But the first peak is always higher than the third+ (assuming the second corresponds to normal 2). That's all.

ADD REPLYlink written 3.7 years ago by markus.riester510

cpad0112, There is another support for that from the BubbleTree plots (generated using this R package). The x-axis presents the copy number ratio relative to the normal sample. The y-axis, HDS, is related to LOH events. It depicts the deviation from heterozygousity of SNPs in the tumor. So the peak (cluster of bubbles) with the lower copy number ratio values also shows extensive LOH. So it makes sense it represents only 1 copy of the chromosome.

ADD REPLYlink written 3.7 years ago by gil.hornung70

Thank you, Markus.

ADD REPLYlink written 3.7 years ago by gil.hornung70
gravatar for Eric T.
3.7 years ago by
Eric T.2.6k
San Francisco, CA
Eric T.2.6k wrote:

Markus' explanation is correct.

To help address this issue in your data without a full analysis of tumor ploidy and heterogeneity, you can use the command call --center median. In CNVkit 0.8.5, this will take a "majority rules" decision on the appropriate center value by first taking the median log2 value of each chromosome, then the median of those values -- ensuring that at least one chromosome with fairly representative ploidy will be centered with a log2 value of 0.0.

It's still possible for this approach to leave the log2 values centered off from the true neutral value. In the development version of CNVkit (available on GitHub), there is a new option --center-at which lets you specify the log2 value that you have independently determined should be the neutral value.

ADD COMMENTlink written 3.7 years ago by Eric T.2.6k
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